In order to maintain their shape and integrity, it is critical that all types of cells contain a structural scaffold. This structure is known as the cytoskeleton and is composed of a framework of interlocking proteins such as microtubules, actin and intermediate filaments. It is currently believed that the controlled regulation of the assembly and disassembly of the cytoskeleton is critical to the survival of the cell and many cellular processes are mediated by the cytoskeleton, especially those involving the interaction of the cell with the surrounding environment. These processes include but are not limited to cell adhesion, motility, and polarity. Cell division or mitosis is also dependent on concerted structural changes in the cytoskeleton.
There are several proteins that, in conjunction with the primary components of the cytoskeleton, act as regulators of cytoskeletal architecture. Microtubule-associated proteins (MAPs) comprise one group of proteins that mediate microtubule assembly and function required for the maintenance of cytoskeletal integrity. MAPs co-purify with microtubule polymers and are defined by their association with the microtubule lattice. These proteins are divided into two classes; motor MAPs which play an integral part in cellular movement, and structural MAPs which dictate the morphologic characteristics of the cell (Maccioni and Cambiazo, Physiol. Rev., 1995, 75, 835-864; Olmsted, Annu. Rev. Cell Biol., 1986, 2, 421-457).
Microtubule-associated protein 4 (also known as MAP4) is a member of the non-neuronal structural MAP family. Studies comparing the bovine, human, and mouse MAP4 sequences demonstrated an 80% similarity among the proteins indicating that they belong to the same family of MAPs (West et al., J. Biol. Chem., 1991, 266, 21886-21896).
Originally isolated from microtubule preparations of differentiated mouse neuroblastoma cells, MAP4 was shown to be encoded by a single gene that expresses multiple transcripts in a tissue-specific manner (Code and Olmsted, Gene, 1992, 122, 367-370; Parysek et al., J. Cell Biol., 1984, 99, 2287-2296). These studies implicate MAP4 in the mediation of processes common to supportive and connective tissue types in the mouse. Further support of this conclusion comes from studies in which a muscle-specific MAP4 transcript was isolated in the mouse and shown to be required for myogenesis (Mangan and Olmsted, Development, 1996, 122, 771-781). In these studies, a plasmid bearing the muscle MAP4 nucleotides 216-1214 in the reverse orientation was transfected into myoblasts. Expression of this construct eliminated muscle MAP4 expression with a concomitant perturbation of myotube formation (Mangan and Olmsted, Development, 1996, 122, 771-781).
MAP4 is believed to affect microtubule dynamics by stabilizing the microtubule lattice (Illenberger et al., J. Biol. Chem., 1996, 271, 10834-10843). It has been shown that this stability is disrupted upon phosphorylation and recently at least two kinases have been reported that phosphorylate MAP4, cdc2 kinase which phosphorylates MAP4 in the M (mitosis) phase of the cell cycle and p110mark kinase (Illenberger et al., J. Biol. Chem., 1996, 271, 10834-10843; Ookata et al., Biochemistry, 1997, 36, 15873-15883).
Overexpression of the full- or partial-length (containing only the microtubule binding domain) MAP4 protein was shown to retard cell growth and inhibit organelle motility and trafficking in vivo (Bulinski et al., J. Cell Sci., 1997, 110, 3055-3064; Nguyen et al., J. Cell Sci., 1997, 110, 281-294). MAP4 expression has been shown to be elevated in cells with mutant p53 oncogene expression and therefore linked to cancer chemotherapeutic drug sensitivity. Immunofluorescent studies of murine fibroblasts transfected with MAP4 revealed that cells overexpressing MAP4 were more sensitive to the cancer drug paclitaxel, and less sensitive to vinca alkaloid treatment (Zhang et al., Oncogene, 1998, 16, 1617-1624).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MAP4. To date, strategies aimed at inhibiting MAP4 function have involved the use of antibodies and antisense expression vectors. Blocking antibodies produced to the microtubule binding domain of MAP4 were used to investigate the role of MAP4 in vivo. Antibodies microinjected into human fibroblasts were shown to sequester MAP4 from microtubule binding. However, these studies suggested that the removal of MAP4 does not affect microtubule assembly, organization, posttranslational modification of tubulin, mitotic progression or microtubule-dependent intracellular organelle distribution which is in contrast to other findings in the art (Wang et al., J. Cell Biol., 1996, 132, 345-357).
Therefore, in light of the contradictions in the literature and the fact that this strategy is untested as a therapeutic protocol, there remains a long felt need for agents capable of effectively inhibiting MAP4 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of MAP4 expression.